Several classes of anemic disorders including a number of relatively common, serious hemolytic conditions are the result of the accumulation of methemoglobin to anomalously high levels in red blood cells. These pathological conditions are the result of an imbalance between two competing processes, the endogenous rate of autoxidation of hemoglobin and its re-reduction by cytochrome b5. This proposal is directed at obtaining basic chemical knowledge directly related to these pathological conditions through the use of a focused array of divergent cutting edge technologies; novel multidimensional heteronuclear NMR of paramagnetic heme-proteins, recombinant methods involving high yield expression systems and novel electrochemical methods. The novel heteronuclear multidimensional NMR methods proposed are uniquely suited to the study of heme-proteins and more importantly to the study of heme-protein complexes. These methods have recently been applied to the study of the cytochrome b5 - cytochrome c complex, an important model for long-range biological electron transfer. It is our intent to extend these studies to the study of the cytochrome b5 - myoglobin complex as a model for the solution structure of the physiologically relevant cytochrome b5 - hemoglobin complex. Although the structures of the component proteins (cytochrome b5, hemoglobin and myoglobin) are known, the structure of the complex has resisted crystallographic characterization. Modern NMR methods and novel extensions of those methods proposed here offer the unique capability of obtaining constraints on the orientation of these proteins in complex and in solution under physiologically relevant conditions. We also plan to study the mechanism by which heme-proteins control midpoint potential and reorganizational energies. These studies will then enable more quantitative interpretation of the mechanism of accelerated autoxidation or altered rates of re-reduction in natural mutants of hemoglobin. Preliminary measurements of midpoint potentials of cytochrome b5 have been made using thin layer cells. Novel electrochemical methods employing surface modified electrodes should enable direct measurement of electron transfer rates of components and hence allow greater insights into factors effecting reorganizational energy and rates of electron transfer between components of the physiological complex. Finally, a long-range goal coupled to the specific proposals described above, involves site directed drug design directed at alleviating the anemic effects. Once we have obtained enough information regarding protein factors which control midpoint potential, rates of autoxidation, and re-reduction we hope to design novel pharmaceutical agents which can stabilize mutant hemoglobins or alter the midpoint potential of cytochrome b5 to facilitate re-reduction of methemoglobin.